JPS60252684A - Fluorescent assembly - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The subject of the invention is diaminetriacetic acid capable of forming metal chelates. In particular, the present invention forms species-bound fluorescent rare earth metal chelates useful for coupling metal ions to organic species, such as organic target molecules or antibodies, preferably useful for fluorescence analysis techniques. This invention relates to trifunctional ligands that are effective for.

Fluorescence technology is finding increasing use in chemical and biochemical and medical analysis. The fluorescent method is extremely sensitive in nature. These measurement methods can provide at least the sensitivity of radiochemical methods without the risks associated with radiation.

U.S. Patent Nos. 4,150,295 and 4,058,7
No. 32 Meijika (dated April 17, 1979 and 1
977 (197', 8. Elsevier/Nort
h'Ho1land Biomedical Pres.
Chapter s) on pages 67-80 discloses the general concept of fluorescently quantifying non-fluorescent species using fluorophores that have a longer decay lifetime compared to surrounding fluorescers. It's been done. In this method, atomic scale fluorescent tags (fluorophores) are chemically and covalently immobilized onto individual molecules of the species. This species can be the organic target molecule itself, it can still be essentially the same molecule as the target molecule, or it can be an antibody specific for the target molecule. After appropriate procedures depending on the analysis format, the tagged species are excited and their fluorescence measured using the time-gated techniques taught in the three references mentioned above. The amount of target is determined using the magnitude of the observed fluorescence and a previously prepared fluorescence/concentration species curve.

Fluorophores and tag species useful in such determinations ideally have long fluorescence decay lifetimes and retain the ability to fluoresce for the duration of the analysis. For sensitive analyses, the fluorophore and its association with other species must be present at very low concentrations, e.g. in the nanogram/cc range and below, e.g.
)/' It is important that it be stable even in the CG range. For many immunoassays, it is desirable that the fluorescent dye be water-soluble so that it can react with the antibody in an environment that preserves immunoreactivity.

The metal chelates of the present invention include within their scope a group of species-binding ligands that have all of these desirable properties. Furthermore, the metal chelates of the present invention are simple and inexpensive to manufacture.

Stated in its broadest sense, the present invention provides that an organic species (referred to herein as ITJ) having one type of functional group, one of the four carboxyl groups of diaminetetraacetic acid, and essentially only one is bonded to the general formula 1 (R is a covalent bridge with a length of two or more atoms (hereinafter simply referred to as a covalent bond bridge), and L is the deprotonation of the above functional group on the seed molecule T, etc. What (deprotonated)
This means that it is possible to form a stable structure having the shape shown in FIG.

Since T can contain one or more L groups, one or more diaminetriacetic acids can be covalently bonded to this great circle.

These covalently bonded products (the formula is called "species-bound diamine triacid") form stable chelate complexes with various metal ions.
This constitutes another aspect of the invention.

In a preferred embodiment, the metal ion M is an ion of a rare earth metal capable of forming a fluorescent chelate complex. It is.

In a third aspect of the invention, particularly preferred complexes are formed when the activator or flooder, such as salicylic acid, is also present as a ternary complex with the rare earth metal ion and the species-bound diamine trifluoride.

In a fourth aspect, the invention provides organic species T, such as the group -N
-H, -N-H, -0-H or -8-I. Such seed generation is generally carried out by combining a substantial excess of diaminetetrafeudic dianhydride (where R is a covalent bridge of two or more atoms in length) with at least one of the above groups on the seed molecule. 1 of the anhydride group of dianhydride
Contact with the above species under 5 conditions to produce a species-bonded diamine triacid anhydride; hydrolyze the remaining anhydride group to produce a species-bound diamine triacid; and dissolve the species-bound diamine triacid. total formation of a species-bound rare earth chelate by mixing with rare earth metal ions; then, optionally after appropriate procedures depending on the type of analysis in question, flooding this species-bound rare earth chelate with an activator, such as salicylic acid;
measuring the fluorescence intensity of the fluorescent complex; and relating the observed fluorescence intensity to the fluorescence intensity of a known concentration of such fluorescent complex.

In a fifth aspect, the invention provides for forming an antibody having one or more diamine triacid molecules covalently attached thereto; forming a rare earth chelate of the antibody-conjugated triacid; Bind the target to the antibody by exposing the tissue, liquid or solid substrate suspected of containing the target to the antibody; remove excess unbound antibody; and measuring or detecting the fluorescence as an indicator of the amount or presence of the target molecule.

The detailed description of the present invention is divided into the following parts: Bi-bonded Diamine Triacid Compounds, Metal Complexes, Ternary Bonding with Flatders, Preparation Methods, Enrichment, and Analytical Techniques.

Species-bound diamine triacid compound These compounds have the structure shown in general formula I.

In formula (1), R is a covalent bridge of two or more atoms. Two or more atoms refers to atoms in the bridge itself. These atoms j-j: can be substituted with other atoms or groups if desired. The function of R is to covalently bond two spaced apart amine diacetate groups together to form a stable compound with metal ions on the two amine diacetate groups. Therefore, any group that performs this function without interfering with the chelating ability of the amine diacetic acid group can be used as R.

R, carbon-carbon including carbon-oxygen ether bridges, carbon-nitrogen polyalkyl secondary or tertiary amide bridges, and alkylene, cycloalkylene or arylene containing substituents that are all unsubstituted or pendant from the bridge; Preferably it is an optionally substituted 2 to 8 atom covalent bridge selected from bridges. Substituents include, for example, alkyl of 1 to about 10 carbon atoms, 6
Included are aryls of ~10 carbon atoms, aralkyls and alkaryls of 7 to about 14 carbon atoms. Bridges or the aforementioned substituents can also be substituted with carboxyl, carbonyl, ether, carbamate, secondary amide, sulfonate, sulfamate, and the like. Examples of R groups include ethylene, n-propylene, inopropylene, various butylenes including n-butylene, 1- and 2-methylpropylene, 1-propylethylene, 1-cyclohexylethylene, 1-phenylethylene, alkyl 1ai- Phenylethylene and hyfurobylene, l-benzylethylene, 2-amidopropylene, cyclohexyl-1,2-
ene, phenyl-1,3-ene, diethylene-ether-CH2-CH2-0-CH2-C'H2-, triethylenediether-CI(2-CH2-0-CH2-CH
2-O-CH2-CI (2-, etc.) This example is not exhaustive, but merely represents a typical embodiment of the R group.

, R is preferably an unsubstituted alkylene having a length of 2 to 5 carbons, preferably 2 to 4 carbons, because it is easy to manufacture;
Ethylene and propylene are more preferred, with ethylene being the most preferred R.

Species-Bound Diamines of the Invention The IJ acid compounds contain one or more functional groups that are primary or secondary amide nitrogens, ester oxygens or thioester sulfurs. L
Each unit of serves as a covalent link between one unit of the diamine ligand and the seed molecule T.

The choice of L is determined by the nature of the seed molecule as L can be derived from an amine (primary or secondary), hydroxyl or thiol present on the seed molecule.

- In general, amide nitrogens are the preferred L groups, with amide groups (thus derived from primary amine groups on T) being most preferred.

The species-bound diamine triacid compounds of the present invention contain two amine groups, one of which carries two acetate groups, and the other of which carries one acetate group and one acetate/L-T addition. carry things. For simplicity, the three acetate groups are protonated (pro!onated) in the introduction and claims.
) is shown as a shape.

It will be appreciated that in reality, these groups that are weak acids exist in equilibrium between the protonated form shown and the corresponding deprotonated salt form -COO-. The exact proportion of the two forms depends on the pI (and composition) of the environment of use. The protonated forms shown are intended to represent the equilibrium of the two forms that actually exist.

Metal Complexes The species-bound diamine toluates of the present invention are effective chelate-forming ligands. Their ability to form complexes appears to be similar, if not essentially the same, as that of the corresponding non-species-bound diaminetetraacetic acid ligand (eg, ethylenediaminetetraacetic acid-EDTA). Although used in further bonding to the species, the quaternary carbonyl group can serve as a coordination point with the metal ion, and therefore the dissociation constant of the complex formed with the IJ acid (diamine) of the present invention is Very similar to that of the complex obtained with diaminetetraacetic acid in the art (
It seems that she is having LJ.

Examples of metal ions M that can be complexed include radioactive nuclei, otide ions, and non-radioactive metal ions. Although not widely proven, metal ions and EDTA
It appears that virtually all complexes known in the art with and their non-species-bound analogues can be prepared using the species-bound ligands of the present invention. For example, complexes can be formed with M equal to ions of transition metals and rare earth metals of the actinide and lanmenide series. Examples of metal ions represented by M include A, +++, 10++ +++ +++ Am, Cd, Ce, c, +++, crn+
++, ten+ ten++ ++ +++ ++ +++Co
, Co , Cr , Cr , Cu , Dy 1+++
+++ ++ +← 10++ Er%Eu, Fe, Fe%Ga.

+++ ++ +++++++-+-++10Gd 1Kg
, Ho, In, La.

Lu+++, Mn←, Seed+++, Nd+←, NI++,
Pb++, Pd++, Pm+→, Pr+←, Pu+←
, P, +++++, sb+++, so+++, sm+
++, Sm++, Sn+10, Tb+, Th++-
H-1TI+++, Tl+++, Tm+++, ■
＋＋＋＋■＋＋＋＋＋, vo2+, Y++＋, Yb++
+, zn+ten, and zr+++1.

Owing to the range of metal ions that can be incorporated and the wide range and diversity of properties exhibited by such metal ions, the metal ion complexes according to the invention are usefully used in the field of chemical analysis. The presence of a metal ion bound to a species molecule according to the invention can be determined by radioactivity analysis, X-ray scattering or fluorescence depending on the metal ion involved.

NMR'1 can be detected by techniques such as ESR shift. In a preferred embodiment, the metal ion used is a rare earth metal ion complexed with an EDTA type chelating ligand. Among these, terbium, dysprosium, europium, samarium and neodymium are preferred, terbium and europium are more preferred, and terbium is the most preferred rare earth for forming the metal ion M.

The complex formed between the metal ion 2.4 and the species-bound diamine triacid is believed to be a 1:1 equimolar metal:chelate complex. Structurally, it is represented by the structure given below.

Ternary Combinations with Flatders A preferred use of the seed-bonded diamine triacids of the present invention is as chelating ligands in fluorescent rare earth metal complexes. It has been found that the effectiveness of these complexes in fluorescence analysis techniques is substantially enhanced when a third component is present in the complex. This third component, the accelerator, is generally called a "flatder" because it is usually added in large excess. Flooders, also referred to as accelerators or sensitizers, increase the fluorescence excitation efficiency of rare earth chelates and are disclosed in non-species bound rare earth ions and chelates.

ANALYST of Dagnall et al., 92.358-
363, (1967) 1 Heller and W.
asserman, J., CHEM, PHYS,
, 42.

949-955, (1965); Me Car
thy and Wineford'ner.

ANAL, CHEM, , 38.848 (196
6); TALAN'rA of Taketatsu et al.
13, 1081-1087. (1966) ;C
See HEM, Takumi, 529-536, (1966). The descriptions of these literary and effective flutters are hereby incorporated by reference. Although this is not an exhaustive list, there are a number of flooding agents that can be used with the species-bound diamine triacids of the present invention.
nt).

Many of these flutters appear to act by occupying locations on rare earth metal ions not already occupied by coordinating and species-bound diamine triacid ligands. By doing so, the flutters bond tightly with the metal ions, allowing effective energy transfer from the flutters to the rare earth metal.

An example of a complex-forming flutter is 5-sulfosalicylic acid (5
-3SA), 4-aminosalicylic acid, salicylic acid, Ike's salicylic acid, dipicolinic acid, etc. These substances 1 are particularly effective against terbium complexes and their application proves their action.

The complex of species-bound diamine triacid and terbium is v24
It exhibits an excitation band of 0OA. This is a relatively weak 1,A band, and at this wavelength it is not a convenient part of the spectrum. When 5-3SA is added as a flutter,
A strong excitation band in the near UV of 3100 A was observed, which is a much more convenient wavelength from a sample transmission standpoint.

Examples of flooders that are particularly effective against europium include salts such as potassium carbonate, sodium tungstate, etc., and organic substances such as phenanthroline.

For example, 10 to I
Except in the case of Q M (in which case the flutter concentration is the same), the amount of flutter used is very high relative to the number of species-bound diamine triacids.

It is generally preferred to use at least a 10-fold increase in the metal ion or diamine triacid on a molar basis, with a molar excess of 10' to 1010 being more preferred.

These large excesses are required because the stability of Flanders diamine complexes is many times lower than that of complexes between dialkaline earth metals.

f4ooded complex
) are simply formed by adding a flutter to a solution of an already formed rare earth chelate complex. This is typically done just before measuring the fluorescence of the flatder-containing complex. Although not known with certainty, the flutter-containing complex that forms is a species-bound diamine triacid:rare earth metal ion:flatter:
], : It seems to be a 1 mole three-component combination.

Preparation method - lA) which must contain the 0-H group is prepared by the general formula m
It can be simply formed by contacting in the liquid phase with a substantial molar excess of diaminetetraacetic dianhydride shown in a polar neutral liquid organic reaction medium.

Effective reaction media include acetone, dimethylformamide (DMF), dimethyl sulfoxide (DMSO).
) and I-rMPA. Mixtures of these substances can also be used. If the reactive group on the seed molecule is an amine, especially a primary amine, water and a mixed solvent containing water can be used as the solvent. - This contact is carried out under conditions which cause the active group of the species to react with one of the two anhydride groups of the diamine.

When the active group of the seed molecule is an amine, the contact is carried out under mild conditions, such as from about 5°C to about 100°C, preferably from about 10°C to about 75°C.
carried out at a temperature of When the active group of the species is a thiol-spanning hydroxyl: 1. Somewhat more severe conditions e.g. 2
A reddening temperature of 5 to about 150°C is commonly used. in this case,
Temperatures of 35 to about 125°C are preferred. This difference in reactivity can be exploited to perform selective reactions with amine groups and exclude reactions with thiols or hydroxyls, if it is advantageous to do so. Of course, the reaction time used is inversely proportional to the use @1 change.

Generally, times of about 0.5 hours to about 2 days are used, with 1 to about 36 hours being preferred. These times and temperatures are given as an indication. In some cases, for example in the case of highly thermally sensitive or thermally insensitive targets, it may be advantageous to fall outside the exemplary ranges shown here. Generally, the use of lower temperatures within these ranges is preferred as it provides better reaction selectivity between the anhydride and species molecules.

It is essential to use an excess of dianhydride.

A dianhydride:species molecular ratio of at least 4:1 should be used; 9. ! A ratio of 5:1 to about 10:1 is preferred.

The upper limit of this ratio is arbitrarily determined based on economic efficiency. Higher amounts of anhydride than required in this range can be used with good results. Such a mode of operation is considered within the scope of the present invention, but is also believed to be futile since excess anhydride is destroyed by hydrolysis in subsequent operations.

The dianhydrides used in the present invention are materials known in the art (U.S. Pat. No. 3,497,535) or are derived from reacting diaminetetraacetic anhydrides. For example, tetraacid is prepared in the presence of a molar excess of acetic anhydride and a tertiary amine by conventional methods of preparation.
It can be prepared by heating at or around 1 degree (i.e., 80 to 180 degrees C) for a long period of time, for example, 12 to 36 hours.

After attachment of the seed molecule to the dianhydride, the remaining anhydride group is hydrolyzed to the corresponding diacetic acid.

This hydrolysis is facilitated by mixing the coupling reaction mixture with an excess amount of water over the number of moles of anhydride to be hydrolyzed and heating. The appropriate temperature is
from about 30 to about 125"C. Suitable times are from about 1 minute to about 2 pupillary intervals. Preferred temperatures and times are from 40 to about 10"C.
5° C., and 2 to 90 minutes. Alternatively, other moxibustion techniques known in the art for hydrolyzing carboxylic anhydrides to acids (eg, acetic anhydride to acetic acid) can be used.

The stoichiometric excess of anhydride (used to form the species-bound triacid) is separated from the quasi-bound material. Removal of this excess anhydride is most conveniently performed before or after hydrolysis of the anhydride to the acid. If the excess anhydride is not removed, it will react with the metal ions and any flooder if present, allowing accurate measurements of the amount of species-bound diamine triacid to perform the separation. It can be done in any way.

Since the two species differ in size, chromatographic techniques such as gel permeation chromatography, high pressure liquid chromatography, column chromatography (eg using silica gel substrates) or thin layer chromatography can be used. Separation can also be accomplished using membrane techniques, including dialysis and ultrafiltration. Often, the bound material differs from the unbound anhydride or acid in some physical property, such as charge or solubility. Differences in such properties can be used as separation criteria for selective extraction or electrophoresis, for example. Although chromatographic separations are used and generally preferred in the present invention, the exact method used should be determined based on the actual substance and separation in question. The conventionally bound diamine triacid without excess anhydride or acid is then formed into a metal complex.

Hydrolysis produces the desired species-bound diamine triacid of Formula 1. These can be converted to metal-coordination compounds of the formula (2) by contacting the triacid with the desired metal ion. This contact is generally carried out in solution. The amount of metal ion is Approximately 1 mole of ions per mole of triacid is required. Complexes of metal ions and triacids are strong complexes with large stability constants.
It is not necessary to use large excesses of metal ions to drive the complexation reaction, meaning that excess ions (in some cases can interfere with the accuracy of subsequent fluorescence measurements).

The metal complex formation step is generally carried out in an aqueous reaction medium. If desired, a water-containing hydrolysis reaction medium can be suitably used. This means that the metal ions are generally suitably added as water-soluble salts such as halides, nitrates, acetates and the like.

The addition of metal ions is suitably carried out at ambient conditions.

High temperatures are not advantageous for complex formation, and considerably lower temperatures do not provide any advantage.

A temperature range of 5 to about 40°C is generally preferred for convenience.

Rare earth metal ion complex Ω case for fluorescence analysis technique
It is often desirable to add a "flooder" to the metal ion complex, and this flooder addition is generally performed immediately before the fluorescence measurement. it is.

This is done by adding an excess of flutter to a solution of the metal ion complex. This addition is carried out under mild conditions, e.g.
It is carried out at a temperature of ~40°C.

Seed Molecules Seed molecules that can be covalently incorporated into the seed-bound diamine triacid materials and complexes of the present invention are described in the aforementioned U.S. Pat. (cited in the present invention) (described in
It is an organic molecule selected from a "target molecule" as described above, a molecule essentially the same as the "target molecule", and an antibody specific to the "target molecule". These seed molecules contain at least one primary amine, secondary amine, thiol, hydroxyl or hydroxyl group. These active groups can be present naturally on the heel molecule, or can be present on the seed molecule 1 (a covalently attached "spacer").
er) can exist on the go unit. The spacer is useful in minimizing the possibility of interference between the diamine triacid group and the species molecule itself. It can be right-handed in the l/-1 case or when some other substrate recognition step must be involved. A "spacer" can also be used to provide the necessary amine, thiol or hydroxyl active group. "Seed" includes materials with and without added spacers.

The use of spacer and "bridge" molecules to link active molecules to non-active groups, such as substrates, has been developed in the fields of enzyme immobilization, chromatography, etc. For example, U.S. Pat.
, 873.514 and Wilche, F.
See EBS LETT, 33 (1) 70-72. Although these techniques do not describe the precise systems involved in the present invention, they do disclose the advantages derived from a wide range of reagents and spacers, ty = , q Vc.
Although the present invention can be practiced with any species of molecule containing the required amine, thiol or hydroxyl groups, either inherently or through the use of spacers, therapeutic agents, enzymes, hormones, peptides, proteins and lipids may be used. It can be particularly advantageously used for biologically active target molecules including macromolecules, habten, antigens, etc. Such molecules usually have at least one requisite active group and are often difficult or even impossible to analyze or detect due to the minute amounts that may be present in biological systems.

Examples of seed molecules are thus simple organic target molecules with the required functional groups such as lower alkanols such as methanol, ethanol, butanol, hexanol, cyclohexanol, etc.; lower aromatic hydroxy compounds such as 7-enols, co-dinitrophenol and Cresol i lower alkyl and aroma! Amines - ethylamine, diethylamine, butylamine, and inpropylamine; lower thiols - propanethiol and butanethiol Kara, hormones thyroxine, triodothyronine, human growth hormone gonadotropin, gastrointestinal hormones, insulin, etc.; therapeutic drugs such as digoxin, morphone, pro- It can span targets ranging from complex pharmaceutical and biological molecules, including proteins such as inamides, antibodies, etc. In the case of large molecules such as proteins and antibodies, there may be more than one (even a large number) of the required functional sites on each molecule.
7+>
The purpose of the present invention is to demonstrate a wide range of targets that can be bound to cydiaminetriic acid. It does not limit the scope of the invention.

Analytical Techniques As already mentioned, the present invention allows for the covalent attachment of complexes of metal ions to seed molecules or antibodies of seed molecules. Detection of these metal ions by methods known in the art can be used to determine the presence or amount of species molecules. Also, as already mentioned, if the metal ion incorporated is a radionucleotide, radioanalytical techniques can be used to measure the abundance of the species. Alternatively, atomic absorption techniques can be used to measure the amount of metal and therefore the amount of target.

In one preferred embodiment, the metal is a rare earth and the resulting chelate is activated by a flutter or other means to generate fluorescence.

A number of techniques developed in the radioimmunoassay field are generally applicable to fluorescence immunoassays. These techniques include culture techniques, competitive binding methods and separation of bound and free labeled targets. For example, RApxoxMMuNoAssAy AND RELA
TEDTECHNI QUES (J, 1. Tho
According to reu et al. C.V.

Mo5by Co,, St, Louts, Mis
souri, (/P71)) and RADI
OIMMUNOASSAY/lN(:LINICALB
IOCHEMISTRY (edited by C, A, Pa5te
rnaak, Hey-den, London, N
ew Yor$c, Rheine, (/ri7j)
) The main difference between radioimmunoassay and fluorescence immunoassay is the final reading stage. In the former case, radioactive decay is calculated,
In the latter case, fluorescence is excited and measured.

U.S. Patent No. ≠, /J'0,2ri No. 5 and No. ≠, 0! I
The time-gated fluorescence technique disclosed in 'r, '732 is a preferred technique to use because substantially increased sensitivity can be achieved.

In the case of the rare earth chelate tagged fluorescent antibodies of the present invention, they can be used for the analysis of free targets in body fluids or in other preferred applications, as long as they have a unique target or group of targets on their surface. Bio-optical cells or bacteria can be used to detect the presence (or quantify) of specific types of bacteria.

The invention is further illustrated in the following Preparations and Examples. These are illustrative of the invention and are not intended to limit the scope of the invention.

3 μm of ethylenediaminetetraacetic acid (EDTA), rio
A mixture of 65 g of acetic anhydride and too much pyridine
Heat to °C and hold at temperature for 2 μh. The mixture is then cooled and filtered in a glove box. The solid obtained is washed with diethyl ether and dried.

It reaches the theoretical weight tS of EDTA dianhydride. Elemental analysis confirms that this dianhydride is the product obtained.

(b) Propylenediamine 42 U.S. Patent No. 3,
According to the method described in No. 660.31g, λl/J-propylenediaminetetraacetic acid, λl/2y acetic anhydride and 113II pyridine are stirred at 63"Q for 3 hours. The reaction product is Evaporate to dryness under vacuum. The residue is collected, rinsed three times with diethyl ether and dried in a vacuum oven at 100° C. The dry product is the desired dianhydride.

(c) Phenylene/,2-diaminetetraacetic dianhydride U.S. Patent No. 3. p,iay/,3-phenyleidiaminetetraacetic acid, 7 by the method described in ltO,,3y1
2 g of acetic anhydride and 39 g of pyridine are stirred at 6° C. for 2 hours. Cool the mixture and filter. The separated solid is washed with benzene and dried. It is the desired dianhydride.

91J/A, binding thyroxine to EDTA dianhydride The ioomi flask is evacuated and flame dried, followed by 3 cycles of vacuum and dry argon filling. Cool the flask and add EDTA dianhydride made in Jυ1≠339 and j
Fill with ootv L-thyroxine sodium salt pentahydrate. This thyroid hormone is fundamentally important for normal growth and metabolism and is commercially available. Dianhydride:
The molar ratio of hormones is t:i.

Addition of 10 rttl DMF gives a pale yellow solution. Cover the flask with foil and heat to 55°C for 9 hours. A spot test shows that all of the hormones react with the dianhydride to give the following products:

(5) ■1 B, hydration product (4) of residual anhydride is not isolated. Instead, add Hiro's distilled water to the flask and continue heating for additional i,z hours. As a result, the remaining free radicals are hydrolyzed to produce thyroxine-linked ethylenediaminetriacetic acid. The triacid and EDTA produced are recovered as liquid F by a suitable filtration method.

C. EDTA and triacid The crude product of B above was passed through a packed silica gel column using ethanol:water (ri!:J-) as eluent, then ♂O:〃riJ-% aqueous ethanol. Elute with :30% aqueous ammonia. Triacid, aqueous ethanol:
It is eluted with aqueous ammonia and collected as a TLCK pure compound.

From NMR1 elemental analysis and IR constant scanning, the recovered product was found to be the desired thyroxine-bound trie (
C) The yield is proven to be 9%.

D, Formation of a complex between C and a rare earth ion Weigh out a known amount of the thyroxine-bound diamine triacetic acid recovered in step C above, and add several ml of 0. /N Dissolved in NaOH. Within minutes, stoichiometric amounts of rare earth halides (
Specifically, in this example terbium chloride) is added and mixed to form a homogeneous solution containing the desired rare earth (terbium) io7/-←thyroxine-bound diamine myacetic acid l:l complex ■) and non-interfering sodium chloride.

Formation of a Ternary Complex of E, D and Flooder An aliquot of the terbium complex of D above is introduced into 10 vrl of water to give a molar concentration of approximately /θ-8. 10-2 mole Hf of s-sulfosalicylic acid is added to form a terbium:thyroxine-linked diamine triacid:7-rad monocomplex. Approximately 33o
When excited with oX, this complex becomes ! ≠! It exhibits strong fluorescence that can be detected by oX. The application of this fluorescent property to the analysis of thyroxine is shown below.

Ward 1 The preparation of Example 1 is repeated with λ changes.

Europium chloride is used in place of terbium chloride in the process. Phenantron') in place of j-8SA 7
Use as a ladder. This results in the formation of a ternary complex force 3 corresponding to the europium chelate and the terbium complex formed in Example/. The europium complex, when used with the phenanthroline flag, is non-fluorescent and allows the amount of thyroxine present in solution to be determined by the method described below.

Example 3 A, conjugate hydrolysis of cholesterol to EDTA dianhydride A 10 ml round bottom flask is flame dried and then filled with argon. Next, 200my (0,jJmm
ol) recrystallized cholesterol and λ7. (J3
Preparation of r9 mmol) Charge the dianhydride of a). 7
The flask is placed under vacuum, then 3 ml of dry DMF is added and the mixture is kept at 0° C. for 36 hours.

During this period the mixture remains essentially homogeneous and colorless.

Cool it to room temperature and add 3 ml of deionized water with further cooling. A precipitate forms and the heterogeneous mixture
Stir for an hour.

When this mixture is diluted with 10 ml of diethyl ether-acetone (/:+), further precipitate forms.

The mixture is filtered and the solids are washed with 10 ml of an ether-acetone mixture. The liquid is collected, evaporated, and diluted with additional ethanol to generate more solids. Collect this and add to the solids collected earlier. After drying, 3-22
A theoretical amount of the desired N-(ethanol-0-cholesterol)ethylenediaminetriacetic acid is obtained. Analysis by NMR and TLC shows that the product is very pure. .Elemental analysis is consistent with the desired compound.

B. Formation of metal ion complex Add 3 ml of the above substance A (3oW). , /NNaOH
dissolve in Rare earth metal ion (terbium or dysprosium) in approximately stoichiometric amount (1 mole of compound)
4 t) i, oo~/, 0 area mole). As a result, a rare earth metal ion complex of substance A is generated.

When this substance is promoted by the addition of excess flooder,
Shows fluorescent properties useful for fluorescence analysis. This addition of the flooder is carried out in the same manner as in Example 1.

3) P-isopropanol (/mmol) of a simple alkanol is mixed with 10 rnl of dry DMF according to the method of Example 3. 1 mmol of the dianhydride from preparation a) is added and the mixture is kept at 0° C. for 21 hours. As a result, the hydroxyl group of the alkanol reacts with the anhydride and the alkanol is combined in ester form. This ester can be separated from residual anhydride before or after hydrolysis of the anhydride units. Complexes can be formed by mixing the ester with the desired metal ion.

1 yu nest of amines Repeat the procedure in Example ≠ with λ changes.

If, ethylamine is used in place of isopropanol on an equimolar basis. Second, reduce the intensity of the reaction conditions and
Use a temperature of 5°C. Ethylamine adds to the dianhydride in the amide form.

Repeat the coupling of Example Hiro using phenol, ethylthiol and diethylamine instead of the inproper tool used in Example Hiro. In the case of diethylamine, a lower temperature (35°C) is used. Repeated steps yield combined products based on each new starting material.If desired, these products can be further processed to form metal ion complexes, e.g. in an aqueous environment. Repeat the calf pulling of Example 5 with two additional changes: instead of the DMF solvent, use water and
30℃ instead of J' hours? A waiting time used O These mild conditions allow the more active amine groups to react,
The amine binds preferentially to the dianhydride to form the desired triacid. This reaction is the same as the preferential reaction of an amine with acetic anhydride in an aqueous environment (JAC8, All, #1 by Caldwell et al. z, (194L-2)) 0 This is the preferred method for coupling triacids to proteins and antibodies. Example 10 A. Coupling Thyronine to PDTA Dianhydride In a dry flask, add the PDTA dianhydride prepared in Preparation. Essential amino acid thyronine/mm obtained from commercially available sources
The molar ratio of dianhydride to thyronine is 6:l. Add Oml of DMF and mix the resulting solution with J
This mixture is then tested on a TLC plate to ensure that it is free of unreacted thyronine and contains the desired thyronine-bound propylene diamine triacetic anhydride. ◎ B. Hydrolysis, separation and complexation By the general method shown in Example 1, the anhydride is added in situ to
Water splits and the resulting excess PDTA is separated by column chromatography ◎ By separation, the desired thyronine-bound triacetic acid is obtained 0 Divide the solution of the desired triacid into L equal parts ◇ The first part (approx. Q. Estimated to contain Jmmol of triacid) as an aqueous solution!
- Mix with 2 mmole of cobalt chloride. As a result,
Cobalt chelate of thyronine-bound triacetic acid is produced. 17. The second part is prepared as a solution of terbium. -Zm
This results in the formation of a terbium chelate of the triacid.
xlo mmole of Fuller Ider Hiro-amine salicylic acid is added to form the / :/ :/ terbium/triacid/Flooder complex. The third portion is carefully contacted with In111- /- as chloride, Zmmol.・In addition to this, thyronine-bound triacid Inul
Example II The preparation of Example 5 is repeated with one modification: EDTA
Cyclohexylene instead of dianhydride! .

Use cocyaminetetraacetic dianhydride. As a result, ethylamine-bound triacetic acid is produced. This compound is contacted with europium ions to form a europium complex, which is then converted to a fluorescently active complex by addition of a -iog molar excess of 7 rad potassium carbonate. This complex can be detected by fluorescence techniques.

As a first step, we demonstrate the fact that the fluorescence of species-bound diamine triacid-'rare earth metal ion complexes is related to the concentration of the complex.

Serial dilutions of a solution of thyroxine-conjugated diamine triacetic acid complex with terbium (prepared as in Example 1 D) to +1
0'~2. ! Perform at a thyroxine concentration of
Sulfosalicylic acid) 6 approx. 11. Dl+ of j, the fluorescence of each sample is IMMUNOF'L, UORESCENC
E station DRELATED 5TAINING TECHN
IQUES, edited by Knapp et al., 67-♂0-
Elrevier /North Holland B
io-medical Press, Amste
Measurements are made using a time-gated fluorimeter and measurement technique as described in RDAM, /77g. Plot the signal on log-log paper and find that it is a straight line in this range.

The curve can be extrapolated to about 10-2 moles as the limit detection value. The detection limit is the concentration at which the fluorescent signal just equals the signal variation from the blank sample. The signal from the blank sample includes optical multiplier dark noise and residual background signal from the sample container, solvents, buffers, etc. The fact that a linear plot is obtained indicates that the concentration of the tag species can be determined by measuring its fluorescence. be converted into

First, a standard curve is constructed along the line as in radioimmunoassay. Serial dilutions of non-species-bound thyroxine are prepared in thyroxine-free plasma. Thyroxine-free plasma is produced by techniques known in the art, including denaturation of total thyroxine-binding plasma proteins.

The concentrations are /, 2, ≠, r and /le μg/de op
Hr.

A fixed amount of complex-bound thyroxine (D in Example 1) is added to each dilution along with a fixed amount of antibody against thyroxine. This mixture is incubated under normal competitive binding conditions ◇ Then the antibody-bound thyroxine and free thyroxine,
Separation is performed using separation steps known in the art such as ammonium sulfate precipitation, double antibody precipitation, polyethylene glycol precipitation, dextran-charcoal precipitation, column separation, and the like. The bound fractions are then suspended in buffer, a flooder is added, and the fluorescence is measured using the time gate method described above.

Creating a zero curve that plots the observed fluorescence against the concentration of unbound thyroxine in a standard sample shows that at larger concentrations of unbound thyroxine, the bound substance has less ability to compete for the antibody site and the observed It is confirmed that the fluorescence is lower. Next, the thyroxine concentration in the unknown sample is measured using the created curve.

Serum samples containing unknown amounts of thyroxine are analyzed for total thyroxine. First, the sample is acidified to denature the bound proteins and free the thyroxine from its binding. next,
After adjusting the threshold (adjustment), the samples are treated with one example/l) part of thyroxine binding, a standard amount of diamine triacid metal complex, and a standard amount of thyroxine binding as was done in the generation of the standard curve. This mixture is cultured as described above. Separate bound and unbound material. Add flooder and measure fluorescence. The thyroxine concentration in the unknown sample is read from the standard curve based on the observed fluorescence.

In some cases, the target molecule (the molecule to be detected or measured) is an antibody. For example, it may be of medical importance to measure the range of immunoglobidin (IgE) antibodies in human patients suffering from allergies. Patients can have different amounts of IgE antibodies against various allergens, and for appropriate treatment it is important to know the distribution of IgE antibodies in the blood or other body fluids.
To measure the amount of E-antibodies, the allergen in question is covalently bound to a solid substrate using known techniques, and the substrate is exposed to the patient's body fluids. After a suitable incubation period under suitable conditions, the substrate is removed and washed. a representative amount of IgE antibodies that differ by % to the allergen on the solid substrate is present on the substrate;
The O substrate bound to the allergen is exposed to a terbium chelate diamine triate-conjugated antibody against human IgE. This anti-IgE antibody generally targets all subclasses of human IgE (5ub-class
ses). After a second exposure, again under suitable conditions, the solid substrate is again washed and exposed to a 7 ladder, such as sodium tungstate. Next, preferably the time-gated fluoromide described above is used.
A standard curve can be constructed using known art methods for each allergen with a known amount of IgE, and thus, by comparison with the standard curve, unknown amounts in human samples can be determined. The Ign of can be measured.

B. Analysis on Cells or Bacteria Cells or bacteria can have various molecular markers on their surface. These markers, in some cases, can be identified, isolated or purified, and used to generate antibodies specific for the markers. Once bound to a metal complex, it can be used to identify certain cells or bacteria that have markers on their surface. cells or bacteria are exposed to diamine triate metal-conjugated antibodies;
When marker L is present, a relatively high concentration of bound antibody is bound to the surface. These cells are then examined with a fluorescence microscope or a fluorescence cell flow system, and the fluorescence cells or bacteria are calculated for a given volume, thus determining the presence of cells or bacteria quantitatively. Or it turns out qualitatively.

These are just two examples of the many possible analyzes that can be performed using the species-bound diamine triacetic acid complexes of the present invention; other equivalent methods can be used as well.

Applicant's agent Kiyoshi Inomata Continued from page 1 ■Int, CI, 4 Identification code Office reference number 33/)
33 7906-2G

Claims (1)

[Claims] 1. A species-bonded diamine triacetic acid having the following formula and essentially 1:
1 mole V-) metal ions containing metal ions in the complex V-
1. A fluorescent combination comprising: [wherein R is a covalent bridge of two or more atoms in length, T is -NH, -NH, -0-H and -S-
Atsuki seed molecule having a functional group selected from H, L is the above functional group in a deprotonated form that covalently bonds T to the carbonyl carbon of diaminetriacetic acid]. 2. The fluorescent combination according to claim 1, wherein the flutter is a salicite. 3. The fluorescent conjugate according to claim 1, wherein the fluorophore is 5-sulfosalicylic acid. 4. A method for producing fluorescent species-bonded diamine triacetic acid, which comprises the following steps a to θ. a, -N-H, -N-H, -0-H, and -8-H per mole of the seed molecule, at least 4 moles of the following formula [wherein R is 2 or more] A step of contacting the diamine dianhydride of the above atomic length covalent bond bridge Ruru] in a liquid phase at a temperature of about 5 to about 100 C for about 0.5 hours to about 2 days, hydrolyzing the residual anhydride groups wK a step of recovering the species-bonded diamine triacetic acid produced in the C5 process; and e. contacting the fluorescent complex with a substantial molar excess of Flooder. 5. A method for fluorescent analysis of the fluorescent combination according to claim 1, which comprises the following steps a and b. a. exciting said fluorescent combination with at least one radiation pulse, said pulse having a short pulse duration compared to the fluorescence decay lifetime of said fluorescent combination; and Measuring the fluorescence of the fluorescent combination after the material fluorescence has substantially decayed. 6. The fluorescent combination is thyroxine-conjugated diamine triacetate Ml
-f Rubiram-5-sulfosalicylic acid complex, the radiation has a wavelength of about 3300 A, and the detection is about 545 A
6. The method according to claim 5, carried out at a wavelength of 0A.